Phosphorus (P) is an essential element for growth. A substantial amount of the phosphorus found in conventional livestock feed, e.g., cereal grains, oil seed meal, and by products that originate from seeds, is in the form of phosphate which is covalently bound in a molecule known as phytate. The bioavailability of phosphorus in this form is generally quite low for non-ruminants, such as poultry and swine, because they lack digestive enzymes for separating phosphorus from the phytate molecule.
Several important consequences of the inability of non-ruminants to utilize phytate may be noted. For example, expense is incurred when inorganic phosphorus (e.g., dicalcium phosphate, defluorinated phosphate) or animal products (e.g., meat and bone meal, fish meal) are added to meet the animals' nutritional requirements for phosphorus. Additionally, phytate can bind or chelate a number of minerals (e.g., calcium, zinc, iron, magnesium, and copper) in the gastrointestinal tract, thereby rendering them unavailable for absorption. Furthermore, most of the phytate present in feed passes through the gastrointestinal tract, elevating the amount of phosphorus in manure. This leads to an increased ecological phosphorus burden on the environment.
Microbial phytase, as a feed additive, has been found to improve the bioavailability of phytate phosphorus in typical non-ruminant diets (See, e.g., Cromwell, et al, 1993). The result is a decreased need to add inorganic phosphorus to animal feeds, as well as lower phosphorus levels in the excreted manure (See, e.g., Kornegay, et al, 1996). In addition to a feed additive, phytases may be used for the production of low-phytin feed fractions. For example, phytases may be used in wet milling of grains for the production of e.g., low-phytin corn steep liquor and low-phytin corn gluten or in a dry milling process in combination with starch hydrolyzing enzymes for the production of glucose and alcohols (e.g., ethanol).
Despite the advantage of using phytases in these applications, surprisingly few known phytases have gained widespread acceptance in the feed, starch liquefaction and alcohol fermentation industries. The reasons for this vary from enzyme to enzyme. Typical concerns relate to high manufacturing costs and/or poor stability/activity of the enzyme in the environment of the desired application. A number of enzymatic criteria must be fulfilled by a phytase if it is to be attractive for widespread use in industrial applications. The more important enzymatic criteria include a high overall specific activity, a low pH optimum, resistance to gastrointestinal proteases and thermostability.
Thermostability is one of the most important prerequisites for successful application of phytase as a feed enzyme and for use in starch liquefaction processes because the phytase in the feed and/or processes are exposed to elevated temperatures. For example, in feed pelleting processes the temperatures are between 60 and 95° C. and in starch liquefaction processes the temperatures are between 75 to 120° C.
The DNA sequence of a Buttiauxella sp P1-29 gene which encodes a phytase was reported in WO 06/043178, published Apr. 27, 2006. Reference is made to SEQ ID NO: 1 and SEQ ID NO:2 and the amino acid sequence of the phytase gene of Buttiauxella sp P1-29 (SEQ ID NO:3) reported therein. Based on various intrinsic properties, the Buttiauxella sp P1-29 phytase represented an excellent starting point from which to begin a mutagenesis program for a thermostable phytase for various commercial applications. WO 06/043178 discloses numerous variants of the Buttiauxella sp P1-29 phytase (see, e.g., Table 1). At least one variant disclosed in WO 06/043178 and designated herein as BP-11 has been further modified. The present invention is directed to variants having altered properties, such as improved properties, including but not limited to a) improved thermostability, b) increased specific activity, and/or c) increased specific activity with retention of thermostability as compared to Buttiauxella sp P1-29 phytase or the BP-11 variant.